2型糖尿病骨质疏松大鼠骨密度与生物力学关系
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摘要
目的:近年来随着人民生活水平迅速提高及人均寿命延长,2型糖尿病患病率逐年升高,由其所致骨质疏松症也随之增加。绝经后骨质疏松症是绝经后妇女高发病,我国60岁以上老年女性骨质疏松症发生率为90.48%。骨质疏松症是一种以骨量减少、骨组织微结构破坏为特征,导致骨脆性增加和易于骨折的代谢性骨病。骨折是骨质疏松症的严重并发症,预防骨质疏松性骨折发生是骨质疏松症治疗的根本目的。通过双能X线骨密度仪(DEXA)测量骨密度是目前诊断骨质疏松症金标准。但骨密度变化仅仅反映骨矿含量多少,骨组织和超微结构变化即骨强度(骨质量)与骨折发生更为密切。骨质疏松时骨量减少与骨结构改变均可直接影响骨生物力学,使骨强度降低,因此测定骨生物力学性能变化可作为评价骨质疏松骨强度的一个重要指标。然而,活体情况下难以测定骨的生物力学性能,应用双能X线骨密度仪测定骨密度仍然是临床骨质疏松诊断及观察疗效的重要手段。文献报道骨密度和骨生物力学性能存在相关性,但目前尚无定论。另有研究报道,2型糖尿病可引起骨量减少、不变或增加,本研究旨在建立2型糖尿病骨质疏松大鼠模型基础上,模拟绝经后和2型糖尿病骨质丢失状态,观察不同状态、不同时间骨密度与生物力学指标的关系,了解2型糖尿病骨密度变化,并进一步阐明骨密度用于评价骨脆性和预测骨折危险性的应用价值。
方法:2.5-3月龄雌性Wistar大鼠100只,适应性喂养1周后随机分为正常对照组(NC组)24只、正常去卵巢组(NOVX组)26只、2型糖尿病组(DC组)24只、2型糖尿病去卵巢组(DOVX组)26只。组间大鼠体重无统计学差异。实验期间NC组和NOVX组常规饲料,DC组和DOVX组大鼠高糖高脂饲料(常规饲料中加入20%蔗糖、15%熟猪油、2.5%胆固醇)喂养8周,禁食12小时后左下腹腔给予STZ(STZ,0.1mmol/L,柠檬酸钠配制,pH4.2)30mg/kg注射。于实验第9周末选取尾静脉空腹血糖(FBG)≥7.8mmol/L且伴有胰岛素抵抗者作为T2DM动物模型,T2DM动物模型复制成功1周后无菌条件下NOVX组和DOVX组大鼠麻醉后行双侧卵巢切除手术。实验共持续22周。实验期间所有大鼠均自由进食水。第0、2、4、6、8、10、12周末各组大鼠测体重、血糖、胰岛素;所有大鼠于成模后0、2、4、6、8、10、12周末应用双能X线骨密度仪,采用小动物骨密度测定软件测定腰椎(L3-L6)、双侧股骨骨密度(Bone mineral density,BMD);成模后0周、4周、8周、12周末处死部分大鼠,取第3腰椎和右股骨进行生物力学实验。大鼠骨密度测定:10%水和氯醛腹腔注射麻醉大鼠,待大鼠处于昏睡状态,四肢肌肉松弛,仰卧位置于DEXA探头之下,固定四肢,使椎体保持竖直,双侧股骨保持水平状态,测定腰椎(L3-L6)、双侧股骨骨密度(BMD)。生物力学标本制备:处死大鼠后,完整取出右股骨、腰椎(L3-L6连续的椎体),剔除表面肌肉,置于4%甲醛中保存。实验前取出,牙托粉固定后,在生物力学试验机上进行最大应力及最大压缩力测定。所有实验数据均采用均数±标准差(x±SD)表示,计量资料多组间比较采用方差分析或秩检验,p<0.05认为有统计学意义。
结果:
1不同时间各组大鼠体重、血糖比较结果
1.1体重比较
NOVX组、NC组大鼠体重均呈增加趋势,且NOVX组大鼠因去卵巢体重增加明显,DOVX组大鼠体重无明显变化,DC组大鼠因糖尿病体重呈减轻趋势。
0周NC与DC、DOVX组两两之间无明显差异(p>0.05),NC与NOVX之间无显著差异(p>0.05),NOVX组与DC、DOVX组之间有显著差异(p<0.05)。2周NOVX体重均显著高于其他各组(p<0.05),NC、DC、DOVX组两两之间无显著差异(p>0.05)。4周DC组大鼠体重最低且与NC、NOVX组有显著差异(p<0.05),NOVX组大鼠体重均高于其他各组且有显著差异(p<0.05)。6与8周任意两组之间比较均有显著差异(p<0.05),DC组大鼠体重最低,NOVX组大鼠体重最高。10周DC组大鼠体重均低于其余各组且有显著差异(p<0.05),NOVX组大鼠体重均高于其余各组且有显著差异(p<0.05)。12周NOVX组与其余各组之间比较均有显著差异(p<0.05),DC组与NC组之间有显著差异(p<0.05),而DOVX与DC、NC组之间无显著差异(p>0.05)。
1.2血糖比较结果
0周糖尿病组(DC、DOVX)FBG显著高于非糖尿病组(NC、NOVX)(p<0.05)。2周DC组、DOVX组FBG出现下降趋势,DC组FBG显著高于NC组、NOVX组(p<0.05),DOVX组FBG显著高于NOVX组(p<0.05),NC组FBG与NOVX组相比无显著差异(p>0.05),DOVX组FBG高于NC组但无显著差异(p>0.05)。4周DC组、DOVX组FBG继续呈下降趋势,且各组之间FBG无显著差异(p>0.05)。6周DC组、DOVX组FBG显著高于NC组、NOVX组(p<0.05),NC组FBG与NOVX组、DC组FBG与DOVX组之间均无显著差异(p>0.05)。8周DC组、DOVX组FBG显著高于NC组、NOVX组(p<0.05),FBG在NC组与NOVX组、DC组与DOVX组之间均无显著差异(p>0.05)。10周各组之间FBG均无显著差异(p>0.05)。12周各组之间FBG均无显著差异(p>0.05)。
1.3胰岛素比较结果
0周:糖尿病组(DC、DOVX)显著高于非糖尿病组(NC、NOVX) (p<0.05)。2周:DC组显著高于NC、NOVX、DOVX组(p<0.05),NOVX组显著低于其他三组(p<0.05)。DOVX组与NC组之间无显著差异(p>0.05)。4周:DC组显著高于NC、NOVX、DOVX组(p<0.05),其余各组两两之间比较无显著差异(p>0.05)。6周:各组之间两两比较无显著差异(p>0.05)。8周:DC、DOVX组显著高于NOVX组(p<0.05),其余各组两两之间比较无显著差异(p>0.05)。10周与12周:各组之间两两比较无显著差异(p>0.05)。
2不同时间大鼠骨密度、生物力学指标比较结果
2.1大鼠骨密度比较结果
2.1.1腰椎骨密度
0周任意两组间比较均无显著性差异,具有可比性。2周任意两组间比较仍无显著性差异(p>0.05)。4周NOVX组和DOVX组明显低于NC组和DC组(p<0.01),NOVX组和DOVX组、NC组和DC组比较无显著性差异(p>0.05)。6周NOVX组、DOVX组和DC组明显低于NC组(p<0.01),NOVX组、DOVX组和DC组之间两两比较无显著性差异(p>0.05)。8周NOVX组、DOVX组和DC组明显低于NC组(p<0.01),NOVX组和DOVX组明显低于DC组(p<0.01),NOVX组和DOVX组之间无显著差异。10周NOVX组、DOVX组和DC组明显低于NC组(p<0.01),NOVX组和DOVX组明显低于DC组(p<0.01),NOVX组和DOVX组之间无显著差异(p>0.05)。12周NOVX组、DOVX组和DC组均明显低于NC组(p<0.01),其中DOVX组降低最为显著,NOVX组、DC组和DOVX组之间两两比较无显著差异(p>0.05)。
2.1.2双侧股骨骨密度
NC组、NOVX组、DC组、DOVX组两两比较,各组间股骨骨密度在不同时间均无明显差异(p>0.05)。
2.2生物力学指标比较结果
2.2.1腰椎最大压缩力
4周时,NOVX组和DOVX组明显低于NC组和DC组(p<0.01),NOVX组和DOVX组、NC组和DC组之间无显著差异(p>0.05)。8周时,NOVX组和DOVX组明显低于NC组和DC组(p<0.01),NOVX组和DOVX组、NC组和DC组之间无显著差异(p>0.05)12周时,NOVX组、DOVX组和DC组明显低于NC组(p<0.01),DC组明显低于NC组(p<0.01),NOVX组和DOVX组又明显低于DC组(p<0.01),NOVX组和DOVX组之间无显著差异(p>0.05)。
2.2.2右股骨最大应力
NC组、NOVX组、DC组、DOVX组两两比较,各组间右股骨最大应力在不同时间均无显著差异(p>0.05)。
3相关分析结果
3.1骨密度与生物力学指标相关分析结果
腰椎最大压缩力与腰椎骨密度相关分析在各组均呈正相关(p<0.01):NC组(r=0.737,p=0.002),NOVX组(r=0.784,p=0.001),DC组(r=0.691,p=0.004),DOVX组(r=0.653,p=0.008)。右股骨最大应力与右股骨骨密度相关分析在各组均无相关性(p>0.05:NC组(r=0.05,p=0.86),NOVX组(r=0.273,p=0.477),DC组r=-0.012,p=0.968),DOVX组(r=0.156,p=0.667)。
3.2骨密度、生物力学指标与血糖相关分析结果
腰椎骨密度与血糖相关分析在各组均无相关性:NC组(r=0.115,p=0.293)(p>0.05) ; NOVX组(r=0.18,p=0.087)(p>0.05) ; DC组(r=0.204,p=0.073)(p>0.05);DOVX组(r=0.059,p=0.569)(p>0.05)。右股骨骨密度与血糖相关分析在各组均无相关性:NC组(r=-0.115,p=0.314)(p>0.05);NOVX组(r=-0.062,p=0.561)(p>0.01);DC组(r=-0.103,p=0.374)(p>0.05);DOVX组(r=0.007,p=0.942)(p>0.05)。左股骨骨密度与血糖相关分析在各组均无相关性:NC组(r=-0.037,p=0.752)(p>0.05);NOVX组(r=-0.035,p=0.752)(p>0.05);DC组(r=-0.065,p=0.598)(p>0.05);DOVX组(r=0.043,p=0.695)(p>0.05)。
腰椎最大压缩力与血糖相关分析在各组均无相关性:NC组(r=-0.041,p=0.884)(p>0.05);NOVX组(r=-0.115,p=0.683)(p>0.05);DC组(r=-0.015,p=0.956)(p>0.05);DOVX组(r=0.374,p=0.208)(p>0.05)。
右股骨最大应力与血糖相关分析在各组均无相关性:NC组(r=0.153,p=0.586)(p>0.05) ; NOVX组(r=0.177,p=0.648)(p>0.05) ; DC组(r=0.019,p=0.952)(p>0.05);DOVX组(r=0.445,p=0.198)(p>0.05)。
结论:
1 2型糖尿病和切除卵巢均促进骨量丢失,导致骨密度下降,主要表现在以松质骨构成为主的椎体。
2 2型糖尿病骨质疏松发生发展过程中,随骨密度下降,生物力学指标也逐渐下降,腰椎最大压缩力与腰椎骨密度呈正相关;骨量减少是骨强度下降部分原因,骨密度可反映骨强度变化。
3空腹血糖与腰椎骨密度无相关性,空腹血糖与腰椎最大压缩力在各组均无相关性。
4右侧股骨最大应力与骨密度无相关性。
5空腹血糖与双侧股骨骨密度无相关性,空腹血糖与右股骨最大应力在各组均无相关性。
Objective:In recent years, with the improvement of people's life quality and extended average life span, the rate of type 2 diabetes increased rapidly, which lead to increased osteoporosis. Postmenpausal women have high risk of osteoporosis. In china, 90.48% of over than 60 years old women have postmenopausal osteoporosis. Osteoporosis is characterized by low bone mass, increased fragility, decreased bone quality, and an increased fracture risk. Fracture is a serious complication of osteoporosis. Therefore, prevent the occurrence of osteoporotic fracture is the main purpose for osteoporosis treatment. Bone mineral density measured by Dual-energy X-ray absorptiometry(DEXA) is the gold standard for osteoporosis diagnose. However, bone mineral density only can reflect the amount of bone mineral concent, bone tissue and ultra-structure is more correlated with bone strength(bone quality) and racture. Both Osteopenia and bone structure alteration may affect bone biomechanics, which leads to decreased bone strength. Therefore, determination of the bone biomechanics marker is important for osteoporosis and bone strength. However, since it is difficult to determine the bone biomechanics index in vivo, bone mineral density measured by dual-energy X-ray absorptiometry is still considered to be an important method for clinical diagnosis and observation. The relationship between bone mineral density and bone biomechanics index still remains inconclusive. Reduced, unchanged or even increased bone density induced by type 2 diabetes have all been reported recently. In this study, we established the type 2 diabetic osteoporosis model in rats mimicking bone loss in postmenopausal and in type 2 diabetes, bone mineral density and biomechanics index at different times and different conditions will be discussed. The alteration of bone mineral density in type 2 diabetes will be investigated. Further, the value of bone mineral density in evaluation of bone fragility and prediction of fracture risk will also be clarified.
Methods: 100 Wistar rats were randomly divided into four groups after fed normal feedstuff for 1 week: normal contorl(NC,N=24) group, normal ovariectomy(NOVX,N=26) group, diabetes contorl(DC,N=24) group, diabetes and ovariectomy(DOVX,N=26) group. Rats in NC group and NOVX group were fed with normal diet while DC group and DOVX group were fed with high-sugar-fat diet(mixed with 20% sugar, 2.5% cholesterin, 15% cooked lard) for 8 weeks, injectde T2DM were made by intraperitoneal injection of STZ(0.1mmol/L, prepared by citrate sodium, pH4.2, and 30mg/Kg) after 12 hours fasting. At the end of 9th weeks, rats with fasting blood glucose(FBG)≥7.8mmol/L and with insulin resistance were considered T2DM. Rats choosed for NOVX and DOVX group were ovariectomied under anesthesia 1 week later after T2DM model were done. This study takes 22 weeks. And all rats were allowed free access to food and water. At 0, 2, 4, 6, 8, 10, and 12 weeks after ovariectomy, body weight and fasting blood glucose were measured. my, bone mineral density of lumbar spine(3-6) and double femur were also measured by dual-energy X-ray absorptiometry by using small animals software at the same time points. At 0, 4, 8 and also 12 weeks, 3 rats chosen from each groups were killed and L3 and right femur were taken for biomechanical measurment. For the bone mineral density measurement: rats were anesthesised by intraperitoneal injection with 10% chloral hydrate and then lying under DEXA detector, limbs were fixed and make lumbar spine straight. Double femur maintained the plane state. Bone mineral density of lumbar spine(3-6) and double femur were measured. Biomechanics sample preparation: When rats were killed, right femur and lumbar spine (L3-L6 vertebral-continuous) were taken completely and muscles were removed. Samples were fixed with dental base acrylic resin powder and maximum stress and maximum compression strength were determined by the biomechanical testing machine, data were presented as mean±standard deviation and variance analysis or nonparametric test were used to test differences between groups by SPSS 11.5. p<0.05 was considered statistically significant.
Results:
1 Body weight and blood glucose at different times
1.1 Body weight
The body weight of NOVX and NC group were increased, the NOVX group increased more due to ovariectomy. No change in body weight in DOVX group. The body weight of DC group was reduced because of high glucose.
0 week: There are no difference found in body weight among NC, DC and DOVX groups(p>0.05). There no difference found in body weight between NC and NOVX groups(p>0.05). There were statistical differences found in body weight among NOVX group, DC group and DOVX group(p<0.05). 2 week: There were no statistical differences found in body weight among NC, DC and DOVX groups(p<0.05). Rats in NOVX group had significantly higher body weight than the other groups(p<0.05). 4 week: Rats in DC group had significantly lower body weight than those in NC and NOVX groups(p<0.05), while NOVX group had significantly higher body weights than other groups(p<0.05). 6 and 8 weeks: Our results had shown that there are differences between any two groups(p<0.05). The body weight of DC group was lowest while NOVX was highest. 10 week: The body weight in DC group was significantly lower than other groups(p<0.05), while NOVX group had significantly higher body weight than other groups(p<0.05). 12 week: It was found that rats in NOVX group had significantly higher body weight than other groups(p<0.05). There were significant differences between DC and NC groups(p<0.05).
1.2 Fasting blood glucose
0 week: The FBG of diabetic rats(DC,DOVX) was significantly higher than non-diabetic rats(NC,NOVX)(p<0.05). 2 week: The FBG of DC and DOVX groups was decreased. The FBG of rats in DC group was significantly higher than NC and NOVX groups(p<0.05). There were significant differences found in FBG levels between DOVX and NOVX groups(p<0.05). Rats in DC group had significantly higher FBG than DOVX group(p<0.05). There were no differences found in FBG between NC and NOVX groups(p>0.05). The FBG of DOVX seems higher than NC, but there were no statistical differences(p>0.05). 4 week: There no differences in FBG found in all groups(p>0.05). 6 week: The FBG of diabetic rats was significantly higher than non-diabetic rats(p<0.05). There were no differences found in FBG between NC and NOVX groups(P>0.05). Neither between DC and DOVX groups(p>0.05). 8 week: The FBG of DC and DOVX groups was significantly higher than that of NC and NOVX groups(p<0.05). There were no differences found in FBG between NC and NOVX(p>0.05), neither between rats in DC and DOVX groups(p>0.05). 10 week and 12 week: There were no differences found in FBG in all groups(p>0.05).
1.3 Plasms insulin level
0 week: Plasma insulin level was higher than in DOVX rat scompared with that in NC group(p<0.05). No differences found in insulin level in other groups(p>0.05). 2 week: It was found that DC group had significantly higher insulin level than NC, NOVX and DOVX groups(p<0.05). NOVX group animals had significantly lower insulin level than other groups(p<0.05). There were no differences found in insulin between NC and DOVX group(p>0.05). 4 week: Rats in DC group had significantly higher insulin than NC, NOVX and DOVX groups(p<0.05). No differences found between other two groups(p>0.05). 6 week: There were on differences found in insulin among all groups(p>0.05). 8 week: It was found that DC and DOVX group had significantly higher insulin levels than NC and NOVX groups(p<0.05). 10 week and 12 week: There were on differences found in insulin among all groups(p>0.05).
2 Bone mineral density and biomechanics index
2.1 Bone mineral density
2.1.1 The BMD of the lumbar spine
0 week and 2 week: There are no significantly difference found in the BMD of the lumbar spine among NC, NOVX, DC and DOVX groups(p>0.05). 4 week: BMD in NOVX group and DOVX group were significantly deceased than NC group and DC group(p<0.01), but there are no significantly difference found in NOVX group and DOVX group, NC group and DC group(p>0.05). 6 week: BMD in NOVX,DOVX and DC group were significantly lower than that of NC group(p<0.01), there are no significantly difference found in NOVX group, DOVX group and DC group(p>0.05). 8 week: NOVX group, DOVX group and DC group had significantly deceased BMD than NC group(p<0.01), while BMD in NOVX and DOVX group were significantly lower than DC group(p<0.01), there are no significantly difference found in NOVX group and DOVX group(p>0.05). 10 week: Rats in NOVX group, DOVX group and DC group has significantly deceased BMD than NC group(p<0.01), NOVX group and DOVX group were significantly deceased than DC group(p<0.01), there are no significantly difference found between NOVX group and DOVX group(p>0.05). 12 week: NOVX group,DOVX group and DC group were significantly lower than NC group(p<0.01), DOVX group was significantly lower, there are no significantly difference found in NOVX group, DC group and DOVX group(p>0.05).
2.1.2 The BMD of femur
There were no significant diference in femur BMD at any time point and between any groups(p>0.05).
2.2 The biomechanics index
2.2.1 Maximum compressive force of the lumbar spine
4 week 8 week: NOVX group and DOVX group were significantly deceased than NC group(p<0.01), while there are no significantly difference found in NOVX group and DOVX group、DC group and NC group(p>0.05).12 week: NOVX group、DOVX group and DC group were significantly lower than NC group(p<0.01), DC group were significantly deceased than NC group(p<0.01), NOVX group and DOVX group were significantly deceased than DC group(p<0.01). There are no significantly difference found in NOVX group and DOVX group(p>0.05).
2.2.2 Maximum stress of right femur
There were no significant difference found in maximum stress of right femur at any time point and between any groups(p>0.05).
3 Results of correlate analysis
3.1 The correlation between BMD and biomechanics index.
There were positive correlation between the BMD and maximum compressive force of the lumbar spine in four groups(p<0.01): NC group(r=0.737,p=0.002), NOVX group (r=0.784,p=0.001), DC group (r=0.691,p=0.004), DOVX group (r=0.653,p=0.008). No correlation was found between the BMD and maximum stress force of the right femur (p>0.05);NC group (r=0.05,p=0.86), NOVX group (r=0.273,p=0.477), DC group (r=-0.012,p=0.968), DOVX group (r=0.156,p=0.667).
3.2 Correlation between fasting blood glucose、biomechanics index and bone mineral density.
Fasting blood glucose was not correlated with bone mineral density of lumbar: NC group (r=0.115,p=0.293) (p>0.05), NOVX group (r=0.18,p=0.087) (p>0.05), DC group (r=0.204,p=0.073) (p>0.05), DOVX group (r=0.059,p=0.569) (p>0.05).
Fasting blood glucose also had no correlation with bone mineral density of right femur:NC group (r=-0.115,p=0.314) (p>0.05),NOVX group (r=-0.062,p=0.561) (p>0.05), DC group(r=-0.103,p=0.374) (p>0.05), DOVX group(r=0.007,p=0.942) (p>0.05).
There were no correlations between fasting blood glucose and bone mineral density of left femur: NC group(r=-0.037,p=0.752)(p>0.05), NOVX group (r=-0.035,p=0.752)(p>0.05), DC group (r=-0.065,p=0.598)(p>0.05), DOVX group (r=0.043,p=0.695)(p>0.05).
There were no correlation between the maximum compressive force of the lumbar spine and glucose: NC group (r=-0.041,p=0.884)(p>0.05), NOVX group (r=-0.115,p=0.683)(p>0.05), DC group (r=-0.015,p=0.956)(p>0.05), DOVX group (r=0.374,p=0.208)(p>0.05).
There were also no correlation between the maximum stress of the right femur and glucose: NC group (r=0.153,p=0.586)(p>0.05), NOVX group (r=0.177,p=0.648)(p>0.05), DC group (r=0.019,p=0.952)(p>0.05), DOVX group (r=0.445,p=0.198)(p>0.05).
Conclusions:
1 Type 2 diabetes and the loss of ovary function may induce bone loss, resulting in decreased bone density, mainly in vertebral.
2 With the bone mineral density decreasing, biomechanics index was also decreased in type 2 diabetes osteoporosis process. A positive correlation was found between bone mineral density and maximum compressive force of the lumbar spine. Decreased bone mass is the part of the reason for bone strength reduction, and bone mineral density may reflect changes in bone strength.
3 No correlation was found between fasting blood glucose and bone mineral density of lumbar spine, and even maximum compressive force of lumbar spine.
4 No correlation was found between maximum stress and bone mineral density of right femur.
5 Fasting blood glucose had no correlation with bone mineral density of dural femur and maximum stress of right femur.
方法:2.5-3月龄雌性Wistar大鼠100只,适应性喂养1周后随机分为正常对照组(NC组)24只、正常去卵巢组(NOVX组)26只、2型糖尿病组(DC组)24只、2型糖尿病去卵巢组(DOVX组)26只。组间大鼠体重无统计学差异。实验期间NC组和NOVX组常规饲料,DC组和DOVX组大鼠高糖高脂饲料(常规饲料中加入20%蔗糖、15%熟猪油、2.5%胆固醇)喂养8周,禁食12小时后左下腹腔给予STZ(STZ,0.1mmol/L,柠檬酸钠配制,pH4.2)30mg/kg注射。于实验第9周末选取尾静脉空腹血糖(FBG)≥7.8mmol/L且伴有胰岛素抵抗者作为T2DM动物模型,T2DM动物模型复制成功1周后无菌条件下NOVX组和DOVX组大鼠麻醉后行双侧卵巢切除手术。实验共持续22周。实验期间所有大鼠均自由进食水。第0、2、4、6、8、10、12周末各组大鼠测体重、血糖、胰岛素;所有大鼠于成模后0、2、4、6、8、10、12周末应用双能X线骨密度仪,采用小动物骨密度测定软件测定腰椎(L3-L6)、双侧股骨骨密度(Bone mineral density,BMD);成模后0周、4周、8周、12周末处死部分大鼠,取第3腰椎和右股骨进行生物力学实验。大鼠骨密度测定:10%水和氯醛腹腔注射麻醉大鼠,待大鼠处于昏睡状态,四肢肌肉松弛,仰卧位置于DEXA探头之下,固定四肢,使椎体保持竖直,双侧股骨保持水平状态,测定腰椎(L3-L6)、双侧股骨骨密度(BMD)。生物力学标本制备:处死大鼠后,完整取出右股骨、腰椎(L3-L6连续的椎体),剔除表面肌肉,置于4%甲醛中保存。实验前取出,牙托粉固定后,在生物力学试验机上进行最大应力及最大压缩力测定。所有实验数据均采用均数±标准差(x±SD)表示,计量资料多组间比较采用方差分析或秩检验,p<0.05认为有统计学意义。
结果:
1不同时间各组大鼠体重、血糖比较结果
1.1体重比较
NOVX组、NC组大鼠体重均呈增加趋势,且NOVX组大鼠因去卵巢体重增加明显,DOVX组大鼠体重无明显变化,DC组大鼠因糖尿病体重呈减轻趋势。
0周NC与DC、DOVX组两两之间无明显差异(p>0.05),NC与NOVX之间无显著差异(p>0.05),NOVX组与DC、DOVX组之间有显著差异(p<0.05)。2周NOVX体重均显著高于其他各组(p<0.05),NC、DC、DOVX组两两之间无显著差异(p>0.05)。4周DC组大鼠体重最低且与NC、NOVX组有显著差异(p<0.05),NOVX组大鼠体重均高于其他各组且有显著差异(p<0.05)。6与8周任意两组之间比较均有显著差异(p<0.05),DC组大鼠体重最低,NOVX组大鼠体重最高。10周DC组大鼠体重均低于其余各组且有显著差异(p<0.05),NOVX组大鼠体重均高于其余各组且有显著差异(p<0.05)。12周NOVX组与其余各组之间比较均有显著差异(p<0.05),DC组与NC组之间有显著差异(p<0.05),而DOVX与DC、NC组之间无显著差异(p>0.05)。
1.2血糖比较结果
0周糖尿病组(DC、DOVX)FBG显著高于非糖尿病组(NC、NOVX)(p<0.05)。2周DC组、DOVX组FBG出现下降趋势,DC组FBG显著高于NC组、NOVX组(p<0.05),DOVX组FBG显著高于NOVX组(p<0.05),NC组FBG与NOVX组相比无显著差异(p>0.05),DOVX组FBG高于NC组但无显著差异(p>0.05)。4周DC组、DOVX组FBG继续呈下降趋势,且各组之间FBG无显著差异(p>0.05)。6周DC组、DOVX组FBG显著高于NC组、NOVX组(p<0.05),NC组FBG与NOVX组、DC组FBG与DOVX组之间均无显著差异(p>0.05)。8周DC组、DOVX组FBG显著高于NC组、NOVX组(p<0.05),FBG在NC组与NOVX组、DC组与DOVX组之间均无显著差异(p>0.05)。10周各组之间FBG均无显著差异(p>0.05)。12周各组之间FBG均无显著差异(p>0.05)。
1.3胰岛素比较结果
0周:糖尿病组(DC、DOVX)显著高于非糖尿病组(NC、NOVX) (p<0.05)。2周:DC组显著高于NC、NOVX、DOVX组(p<0.05),NOVX组显著低于其他三组(p<0.05)。DOVX组与NC组之间无显著差异(p>0.05)。4周:DC组显著高于NC、NOVX、DOVX组(p<0.05),其余各组两两之间比较无显著差异(p>0.05)。6周:各组之间两两比较无显著差异(p>0.05)。8周:DC、DOVX组显著高于NOVX组(p<0.05),其余各组两两之间比较无显著差异(p>0.05)。10周与12周:各组之间两两比较无显著差异(p>0.05)。
2不同时间大鼠骨密度、生物力学指标比较结果
2.1大鼠骨密度比较结果
2.1.1腰椎骨密度
0周任意两组间比较均无显著性差异,具有可比性。2周任意两组间比较仍无显著性差异(p>0.05)。4周NOVX组和DOVX组明显低于NC组和DC组(p<0.01),NOVX组和DOVX组、NC组和DC组比较无显著性差异(p>0.05)。6周NOVX组、DOVX组和DC组明显低于NC组(p<0.01),NOVX组、DOVX组和DC组之间两两比较无显著性差异(p>0.05)。8周NOVX组、DOVX组和DC组明显低于NC组(p<0.01),NOVX组和DOVX组明显低于DC组(p<0.01),NOVX组和DOVX组之间无显著差异。10周NOVX组、DOVX组和DC组明显低于NC组(p<0.01),NOVX组和DOVX组明显低于DC组(p<0.01),NOVX组和DOVX组之间无显著差异(p>0.05)。12周NOVX组、DOVX组和DC组均明显低于NC组(p<0.01),其中DOVX组降低最为显著,NOVX组、DC组和DOVX组之间两两比较无显著差异(p>0.05)。
2.1.2双侧股骨骨密度
NC组、NOVX组、DC组、DOVX组两两比较,各组间股骨骨密度在不同时间均无明显差异(p>0.05)。
2.2生物力学指标比较结果
2.2.1腰椎最大压缩力
4周时,NOVX组和DOVX组明显低于NC组和DC组(p<0.01),NOVX组和DOVX组、NC组和DC组之间无显著差异(p>0.05)。8周时,NOVX组和DOVX组明显低于NC组和DC组(p<0.01),NOVX组和DOVX组、NC组和DC组之间无显著差异(p>0.05)12周时,NOVX组、DOVX组和DC组明显低于NC组(p<0.01),DC组明显低于NC组(p<0.01),NOVX组和DOVX组又明显低于DC组(p<0.01),NOVX组和DOVX组之间无显著差异(p>0.05)。
2.2.2右股骨最大应力
NC组、NOVX组、DC组、DOVX组两两比较,各组间右股骨最大应力在不同时间均无显著差异(p>0.05)。
3相关分析结果
3.1骨密度与生物力学指标相关分析结果
腰椎最大压缩力与腰椎骨密度相关分析在各组均呈正相关(p<0.01):NC组(r=0.737,p=0.002),NOVX组(r=0.784,p=0.001),DC组(r=0.691,p=0.004),DOVX组(r=0.653,p=0.008)。右股骨最大应力与右股骨骨密度相关分析在各组均无相关性(p>0.05:NC组(r=0.05,p=0.86),NOVX组(r=0.273,p=0.477),DC组r=-0.012,p=0.968),DOVX组(r=0.156,p=0.667)。
3.2骨密度、生物力学指标与血糖相关分析结果
腰椎骨密度与血糖相关分析在各组均无相关性:NC组(r=0.115,p=0.293)(p>0.05) ; NOVX组(r=0.18,p=0.087)(p>0.05) ; DC组(r=0.204,p=0.073)(p>0.05);DOVX组(r=0.059,p=0.569)(p>0.05)。右股骨骨密度与血糖相关分析在各组均无相关性:NC组(r=-0.115,p=0.314)(p>0.05);NOVX组(r=-0.062,p=0.561)(p>0.01);DC组(r=-0.103,p=0.374)(p>0.05);DOVX组(r=0.007,p=0.942)(p>0.05)。左股骨骨密度与血糖相关分析在各组均无相关性:NC组(r=-0.037,p=0.752)(p>0.05);NOVX组(r=-0.035,p=0.752)(p>0.05);DC组(r=-0.065,p=0.598)(p>0.05);DOVX组(r=0.043,p=0.695)(p>0.05)。
腰椎最大压缩力与血糖相关分析在各组均无相关性:NC组(r=-0.041,p=0.884)(p>0.05);NOVX组(r=-0.115,p=0.683)(p>0.05);DC组(r=-0.015,p=0.956)(p>0.05);DOVX组(r=0.374,p=0.208)(p>0.05)。
右股骨最大应力与血糖相关分析在各组均无相关性:NC组(r=0.153,p=0.586)(p>0.05) ; NOVX组(r=0.177,p=0.648)(p>0.05) ; DC组(r=0.019,p=0.952)(p>0.05);DOVX组(r=0.445,p=0.198)(p>0.05)。
结论:
1 2型糖尿病和切除卵巢均促进骨量丢失,导致骨密度下降,主要表现在以松质骨构成为主的椎体。
2 2型糖尿病骨质疏松发生发展过程中,随骨密度下降,生物力学指标也逐渐下降,腰椎最大压缩力与腰椎骨密度呈正相关;骨量减少是骨强度下降部分原因,骨密度可反映骨强度变化。
3空腹血糖与腰椎骨密度无相关性,空腹血糖与腰椎最大压缩力在各组均无相关性。
4右侧股骨最大应力与骨密度无相关性。
5空腹血糖与双侧股骨骨密度无相关性,空腹血糖与右股骨最大应力在各组均无相关性。
Objective:In recent years, with the improvement of people's life quality and extended average life span, the rate of type 2 diabetes increased rapidly, which lead to increased osteoporosis. Postmenpausal women have high risk of osteoporosis. In china, 90.48% of over than 60 years old women have postmenopausal osteoporosis. Osteoporosis is characterized by low bone mass, increased fragility, decreased bone quality, and an increased fracture risk. Fracture is a serious complication of osteoporosis. Therefore, prevent the occurrence of osteoporotic fracture is the main purpose for osteoporosis treatment. Bone mineral density measured by Dual-energy X-ray absorptiometry(DEXA) is the gold standard for osteoporosis diagnose. However, bone mineral density only can reflect the amount of bone mineral concent, bone tissue and ultra-structure is more correlated with bone strength(bone quality) and racture. Both Osteopenia and bone structure alteration may affect bone biomechanics, which leads to decreased bone strength. Therefore, determination of the bone biomechanics marker is important for osteoporosis and bone strength. However, since it is difficult to determine the bone biomechanics index in vivo, bone mineral density measured by dual-energy X-ray absorptiometry is still considered to be an important method for clinical diagnosis and observation. The relationship between bone mineral density and bone biomechanics index still remains inconclusive. Reduced, unchanged or even increased bone density induced by type 2 diabetes have all been reported recently. In this study, we established the type 2 diabetic osteoporosis model in rats mimicking bone loss in postmenopausal and in type 2 diabetes, bone mineral density and biomechanics index at different times and different conditions will be discussed. The alteration of bone mineral density in type 2 diabetes will be investigated. Further, the value of bone mineral density in evaluation of bone fragility and prediction of fracture risk will also be clarified.
Methods: 100 Wistar rats were randomly divided into four groups after fed normal feedstuff for 1 week: normal contorl(NC,N=24) group, normal ovariectomy(NOVX,N=26) group, diabetes contorl(DC,N=24) group, diabetes and ovariectomy(DOVX,N=26) group. Rats in NC group and NOVX group were fed with normal diet while DC group and DOVX group were fed with high-sugar-fat diet(mixed with 20% sugar, 2.5% cholesterin, 15% cooked lard) for 8 weeks, injectde T2DM were made by intraperitoneal injection of STZ(0.1mmol/L, prepared by citrate sodium, pH4.2, and 30mg/Kg) after 12 hours fasting. At the end of 9th weeks, rats with fasting blood glucose(FBG)≥7.8mmol/L and with insulin resistance were considered T2DM. Rats choosed for NOVX and DOVX group were ovariectomied under anesthesia 1 week later after T2DM model were done. This study takes 22 weeks. And all rats were allowed free access to food and water. At 0, 2, 4, 6, 8, 10, and 12 weeks after ovariectomy, body weight and fasting blood glucose were measured. my, bone mineral density of lumbar spine(3-6) and double femur were also measured by dual-energy X-ray absorptiometry by using small animals software at the same time points. At 0, 4, 8 and also 12 weeks, 3 rats chosen from each groups were killed and L3 and right femur were taken for biomechanical measurment. For the bone mineral density measurement: rats were anesthesised by intraperitoneal injection with 10% chloral hydrate and then lying under DEXA detector, limbs were fixed and make lumbar spine straight. Double femur maintained the plane state. Bone mineral density of lumbar spine(3-6) and double femur were measured. Biomechanics sample preparation: When rats were killed, right femur and lumbar spine (L3-L6 vertebral-continuous) were taken completely and muscles were removed. Samples were fixed with dental base acrylic resin powder and maximum stress and maximum compression strength were determined by the biomechanical testing machine, data were presented as mean±standard deviation and variance analysis or nonparametric test were used to test differences between groups by SPSS 11.5. p<0.05 was considered statistically significant.
Results:
1 Body weight and blood glucose at different times
1.1 Body weight
The body weight of NOVX and NC group were increased, the NOVX group increased more due to ovariectomy. No change in body weight in DOVX group. The body weight of DC group was reduced because of high glucose.
0 week: There are no difference found in body weight among NC, DC and DOVX groups(p>0.05). There no difference found in body weight between NC and NOVX groups(p>0.05). There were statistical differences found in body weight among NOVX group, DC group and DOVX group(p<0.05). 2 week: There were no statistical differences found in body weight among NC, DC and DOVX groups(p<0.05). Rats in NOVX group had significantly higher body weight than the other groups(p<0.05). 4 week: Rats in DC group had significantly lower body weight than those in NC and NOVX groups(p<0.05), while NOVX group had significantly higher body weights than other groups(p<0.05). 6 and 8 weeks: Our results had shown that there are differences between any two groups(p<0.05). The body weight of DC group was lowest while NOVX was highest. 10 week: The body weight in DC group was significantly lower than other groups(p<0.05), while NOVX group had significantly higher body weight than other groups(p<0.05). 12 week: It was found that rats in NOVX group had significantly higher body weight than other groups(p<0.05). There were significant differences between DC and NC groups(p<0.05).
1.2 Fasting blood glucose
0 week: The FBG of diabetic rats(DC,DOVX) was significantly higher than non-diabetic rats(NC,NOVX)(p<0.05). 2 week: The FBG of DC and DOVX groups was decreased. The FBG of rats in DC group was significantly higher than NC and NOVX groups(p<0.05). There were significant differences found in FBG levels between DOVX and NOVX groups(p<0.05). Rats in DC group had significantly higher FBG than DOVX group(p<0.05). There were no differences found in FBG between NC and NOVX groups(p>0.05). The FBG of DOVX seems higher than NC, but there were no statistical differences(p>0.05). 4 week: There no differences in FBG found in all groups(p>0.05). 6 week: The FBG of diabetic rats was significantly higher than non-diabetic rats(p<0.05). There were no differences found in FBG between NC and NOVX groups(P>0.05). Neither between DC and DOVX groups(p>0.05). 8 week: The FBG of DC and DOVX groups was significantly higher than that of NC and NOVX groups(p<0.05). There were no differences found in FBG between NC and NOVX(p>0.05), neither between rats in DC and DOVX groups(p>0.05). 10 week and 12 week: There were no differences found in FBG in all groups(p>0.05).
1.3 Plasms insulin level
0 week: Plasma insulin level was higher than in DOVX rat scompared with that in NC group(p<0.05). No differences found in insulin level in other groups(p>0.05). 2 week: It was found that DC group had significantly higher insulin level than NC, NOVX and DOVX groups(p<0.05). NOVX group animals had significantly lower insulin level than other groups(p<0.05). There were no differences found in insulin between NC and DOVX group(p>0.05). 4 week: Rats in DC group had significantly higher insulin than NC, NOVX and DOVX groups(p<0.05). No differences found between other two groups(p>0.05). 6 week: There were on differences found in insulin among all groups(p>0.05). 8 week: It was found that DC and DOVX group had significantly higher insulin levels than NC and NOVX groups(p<0.05). 10 week and 12 week: There were on differences found in insulin among all groups(p>0.05).
2 Bone mineral density and biomechanics index
2.1 Bone mineral density
2.1.1 The BMD of the lumbar spine
0 week and 2 week: There are no significantly difference found in the BMD of the lumbar spine among NC, NOVX, DC and DOVX groups(p>0.05). 4 week: BMD in NOVX group and DOVX group were significantly deceased than NC group and DC group(p<0.01), but there are no significantly difference found in NOVX group and DOVX group, NC group and DC group(p>0.05). 6 week: BMD in NOVX,DOVX and DC group were significantly lower than that of NC group(p<0.01), there are no significantly difference found in NOVX group, DOVX group and DC group(p>0.05). 8 week: NOVX group, DOVX group and DC group had significantly deceased BMD than NC group(p<0.01), while BMD in NOVX and DOVX group were significantly lower than DC group(p<0.01), there are no significantly difference found in NOVX group and DOVX group(p>0.05). 10 week: Rats in NOVX group, DOVX group and DC group has significantly deceased BMD than NC group(p<0.01), NOVX group and DOVX group were significantly deceased than DC group(p<0.01), there are no significantly difference found between NOVX group and DOVX group(p>0.05). 12 week: NOVX group,DOVX group and DC group were significantly lower than NC group(p<0.01), DOVX group was significantly lower, there are no significantly difference found in NOVX group, DC group and DOVX group(p>0.05).
2.1.2 The BMD of femur
There were no significant diference in femur BMD at any time point and between any groups(p>0.05).
2.2 The biomechanics index
2.2.1 Maximum compressive force of the lumbar spine
4 week 8 week: NOVX group and DOVX group were significantly deceased than NC group(p<0.01), while there are no significantly difference found in NOVX group and DOVX group、DC group and NC group(p>0.05).12 week: NOVX group、DOVX group and DC group were significantly lower than NC group(p<0.01), DC group were significantly deceased than NC group(p<0.01), NOVX group and DOVX group were significantly deceased than DC group(p<0.01). There are no significantly difference found in NOVX group and DOVX group(p>0.05).
2.2.2 Maximum stress of right femur
There were no significant difference found in maximum stress of right femur at any time point and between any groups(p>0.05).
3 Results of correlate analysis
3.1 The correlation between BMD and biomechanics index.
There were positive correlation between the BMD and maximum compressive force of the lumbar spine in four groups(p<0.01): NC group(r=0.737,p=0.002), NOVX group (r=0.784,p=0.001), DC group (r=0.691,p=0.004), DOVX group (r=0.653,p=0.008). No correlation was found between the BMD and maximum stress force of the right femur (p>0.05);NC group (r=0.05,p=0.86), NOVX group (r=0.273,p=0.477), DC group (r=-0.012,p=0.968), DOVX group (r=0.156,p=0.667).
3.2 Correlation between fasting blood glucose、biomechanics index and bone mineral density.
Fasting blood glucose was not correlated with bone mineral density of lumbar: NC group (r=0.115,p=0.293) (p>0.05), NOVX group (r=0.18,p=0.087) (p>0.05), DC group (r=0.204,p=0.073) (p>0.05), DOVX group (r=0.059,p=0.569) (p>0.05).
Fasting blood glucose also had no correlation with bone mineral density of right femur:NC group (r=-0.115,p=0.314) (p>0.05),NOVX group (r=-0.062,p=0.561) (p>0.05), DC group(r=-0.103,p=0.374) (p>0.05), DOVX group(r=0.007,p=0.942) (p>0.05).
There were no correlations between fasting blood glucose and bone mineral density of left femur: NC group(r=-0.037,p=0.752)(p>0.05), NOVX group (r=-0.035,p=0.752)(p>0.05), DC group (r=-0.065,p=0.598)(p>0.05), DOVX group (r=0.043,p=0.695)(p>0.05).
There were no correlation between the maximum compressive force of the lumbar spine and glucose: NC group (r=-0.041,p=0.884)(p>0.05), NOVX group (r=-0.115,p=0.683)(p>0.05), DC group (r=-0.015,p=0.956)(p>0.05), DOVX group (r=0.374,p=0.208)(p>0.05).
There were also no correlation between the maximum stress of the right femur and glucose: NC group (r=0.153,p=0.586)(p>0.05), NOVX group (r=0.177,p=0.648)(p>0.05), DC group (r=0.019,p=0.952)(p>0.05), DOVX group (r=0.445,p=0.198)(p>0.05).
Conclusions:
1 Type 2 diabetes and the loss of ovary function may induce bone loss, resulting in decreased bone density, mainly in vertebral.
2 With the bone mineral density decreasing, biomechanics index was also decreased in type 2 diabetes osteoporosis process. A positive correlation was found between bone mineral density and maximum compressive force of the lumbar spine. Decreased bone mass is the part of the reason for bone strength reduction, and bone mineral density may reflect changes in bone strength.
3 No correlation was found between fasting blood glucose and bone mineral density of lumbar spine, and even maximum compressive force of lumbar spine.
4 No correlation was found between maximum stress and bone mineral density of right femur.
5 Fasting blood glucose had no correlation with bone mineral density of dural femur and maximum stress of right femur.
引文
1 Peng ZQ,Vaananen HK,Zhang HX,et al.Long-term effects of ovariectomy on the mechanical properties and chemical composition of rat bone,Bone,1997,20(3):207-212
2 Kharode YP,Sharp MC,Mary H,et al.Utility of the ovariectomizedratasa model for human osteoporosis in drug discovery,Methods in molecular biology,2008,45(5):111-124
3 Leitner MM,Tami AE,Hime M,et al.Longitudinal as well as age-matched assessments of bone changes in the mature ovariectomized rat model, Laboratory animals,2009,43(3):266-271
4 Srinivasan K,Ramarao P.Animal models in type 2 diabetes research:an overview,The Indian journal of medical research,2007,125(3):451-472
5 Unnanuntana A,Gladnick BP,Swift JM,et al.The assessment of fracture risk, The journal of bone and joint surgery American volume,2010,92(3):743-753
6 Kennedy OD,Brennan O,Ramarao P,et al.Effects of high bone turnover on the biomechanical properties of the L3 vertebra in an ovine model of early stage osteoporosis,Spine,2008,33(23):2518-2523
7 Kemper A,Ng T,Ma Y,et al.The biomechanical response of human bone: the influence of bone volume and mineral density. Biomedical sciences instrumentation,2006,42:284-289
8 Dai R,Ma Y,Kemper A,Prisby RD,et al.Effects of genistein on vertebral trabecular bone microstructure,bone mineral density,microcracks,osteocyte density,and bone strength in ovariectomized rats,Journal of bone and mineral metabolism,2008,26(4):342-349
9 Prisby RD,Swift JM,Franke S,et al.Altered bone mass,geometry and mechanical properties during the development and progression of type 2 diabetes in the Zucker diabetic fatty rat,J Endocrinol,2008,199(3):379-388
10刘亚平,季虹,荣海钦,等.2型糖尿病雌性大鼠骨代谢的特点,中国骨质疏松杂志,2008,14(1):35-38
11 Lipscombe LL,Jamal SA,Ma GZ,et al.The Risk of Hip Fractures in Older Individuals With Diabetes:A population-based study,diabetes care,2007, 30(4):835-841
12 Hanley DA,Tenenhouse A,Brennan O,et al.Associations among disease conditions,bone mineral density,and prevalent vertebral deformities in men and women 50 years of age and older:cross 2 sectional results from the Canadian Multicentre Osteoporosis Study,J Bone Miner Res,2003,18(4): 784-790
13 Hadjidakis DI,Androulakis II,Sommer M,et al.Diabetes in postmenopause: different influence on bone mass according to age and disease duration, Experimental and clinical endocrinology diabetes, 2009, 117(5):199-204
14 Franke S,Sommer M,Courtland HW,et al.Advanced glycation end products induce cell cycle arrest and proinflammatory changes in osteoarthritic fibroblast-like synovial cells,Arthritis research therapy,2009,1(5):R136
15 TakagiM,Kasayama S,Yamamo T,et al.A dvanced glycation end products stimulate interleukin-6 production by human bone-derived cells,Bone Miner Res,2003,12:439
16 Elis S,Courtland HW,Tenenhouse A,et al.Elevated serum levels of IGF-1 are sufficient to establish normal body size and skeletal properties,even in the absence of tissue IGF-1,Journal of bone and mineral research,2010,14:86-90
17 Kanazawal I,Yamaguchi T,Kasayama S,et al.Serum insulin-like growth factor-1 level is associated with the presence of vertebral fractures in postmenopausal women with type 2 diabetes mellitus,Osteoporosis international,2007,18(12):1675-1681
18秦健.2型糖尿病患者血糖控制前后对部分骨代谢指标、IGF-1及骨密度的影响,青岛医药卫生,2007,39(1):729
19黄昶荃,程刚.胰岛素抵抗对2型糖尿病及单纯胰岛素抵抗老龄大鼠的肾损伤和骨密度的影响,中国老年学杂志,2006,26:496-498
20 Chang-Quan H,Bi-Rong D,Swift JM,et al.Insufficient renal 1-alpha hydroxylase and bone homeostasis in aged rats with insulin resistance ortype 2 diabetes mellitus,Journal of bone and mineral metabolism, 2008, 26(6):561-568
21 Huang CQ,Ma GZ,Ma Y,et al.The relationship among renal injury, changed activity of trnal 1-alpha hydroxylase and bone loss in elderly rats with insulin resistance or type 2 diabetes mellitus. Journal of endocrinological investigation,2009,32(3):196-201
22 Magana JJ,Gomez R,Nabipour I,et al.Association of interleukin-6 gene polymorphisms with bone mineral density in Mexican women,Archives of medical research,2008,39(6):618-624
23 Li Y,Backesjo CM,Su Sc,et al.IL-6 receptor expression and IL-6 effects change during osteoblast differentiation,Cytokine,2008,43(2):165-173
24 Su Sc,Pei D,et al.Circulating pro-inflammatory cytokines and adiponectin in young men with type 2 diabetes,Acta diabetologica,2010,8:467-472
25 Xiang GD,Pu JH,Kono K,et al.Association between plasma osteoprotegerin concentrations and urinary albumin excretion in type 2 diabetes,Diabet med,2009,26(4):397-403
26 Nabipour I,Kalantarhormozi M,Courtland HW,et al.Osteoprotegerin in relation to type 2 diabetes mellitus and the metabolic syndrome in postmenopausal women,Metabolism clinical and experimental,2009, 17 (6):589-595
27 Fujii H,Kono K,Kasayama S,et al.Oxidative and Nitrosative Stress and Progression of Diabetic Nephropathy in type 2 diabetes,American journal of nephrology,2010,31(4):342-352
28 Schwartz AV,Sellmeyer DE,Strotmeyer ES,et al.Diabetes and bone loss at the hip in older black and white adults,J Bone Miner Res,2005,20(4):596-603
29 Janghorbani M,Van Dam RM,Willett WC,et al.Systematic review of type 1 and type 2 diabetes mellitus and risk of fracture,Am J Epidemiol, 2007,166(5):495-505
30 Strotmeyer ES,Cauley JA,Schwartz AV,et al.Diabetes is associated independently of body composition with BMD and bone volume in olderwhite and black men and women:The Health,Aging,and Body Composition Study,J Bone Miner Res,2004,19(7):1084-1091
31郝明,张瑜庆,延爱锦.老年2型糖尿病患者骨密度变化的探讨,山西医药杂志,2009,38(2):160-161
32李国娟,肖自幼,何皓,等.绝经后女性2型糖尿病患者骨密度变化及其相关因素探讨,实用糖尿病杂志,2008,4(2):34-35
33 Reinwald S,Peterson RG,Sellmeyer DE,et al.Skeletal changes associated with the onset of type 2 diabetes in the ZDF and ZDSD rodent models. American journal of physiology,endocrinology and metabolism, 2009,296(4):765-774
34张丽萍,荣海钦,季虹,等.2型糖尿病大鼠骨代谢和骨生物力学的观察,中国骨质疏松杂志,2008,14(5):318-323
1 Li Liang,Ji-feng Yu,Ken V,et al.Effect of estrogen receptor beta on the osteoblastic differentiation function of human periodontal ligament cells,archives of oral biology,2008,53(12):553–557
2李蔚,孙宜萍,朱国英,等.雌激素在体外对大鼠成骨细胞的作用,中国组织工程研究与临床康复,2008,12(11):2061-2064
3 Yan Zhou,Yun Fu,Lin Yang,et al.The role of estrogen in osteogenetic cytokine expression in human periodontal ligament cells,The international journal of periodontics restorative dentistry,2009,29(5):507-509
4 Sun J S,Li YY,Liu MH,et al.Effects of coumestrol on neonatal and adult mice osteoblast s activities,J Biomed Mater Res A,2007,81:214-223
5李蔚,孙宜萍,朱国英.雌激素在体外对大鼠成骨细胞的作用,中国组织工程研究与临床康复,2008,12(11):2061-2064
6武兆忠,刘敏,冯鉴强,等.雌激素膜快速效应对人成骨样细胞MG63OPGmRNA快速表达水平的影响,中国老年学杂志,2009,29 (10):1222-1224
7 Hertrampf T, Schleipen B, Velders M, et al.Estrogen receptor subtype-specific effects on markers of bone homeostasis,Mol cell endocrinol,2008,10,291(1-2):104-8
8 Pantschenko AG,Zhang W,Nahounou M,et al.Effect of osteoblast-targeted expression of bcl-2 in bone:differential response in male and female mice,J Bone Miner Res,2005,20(8):1414-1429
9 Peter G.Bradford,Ken V.Gerace,Toshimi Aizawa,et al. Estrogen regulation of apoptosis in osteoblasts,Physiology behavior,2010,99(56)181-185
10 Omar Khalid,Sanjeev K,Delhon I,et al.Modulation of Runx2 activity by Estrogen Receptorα:Implications for Osteoporosis and Breast Cancer,The Endocrine society,2008,23(6):11-13
11 Maruyama Z,Yoshida CA,Furuichi T,et al.Runx2 determines bonematurity and turnover rate in postnatal bone development and is involved in bone loss in estrogen deficiency,Dev Dyn,2007,236(7):1876-1890
12 Pufe T,Claassen H,Scholz-Ahrens KE,et al.Influence of estradiol on vascular endothelial growth factor expression in bone:a study in Gottingen miniature pigs and human osteoblasts,Calcif Tissue Int,2007, 80(3):184-191
13 Hiroyuki Takano,Toshimi Aizawa,Sanjeev K,et al.Normal bone growth requires optimal estrogen levels:negative effects of both high and low dose estrogen on the number of growth plate chondrocytes,Tohoku J Exp, 2008,214(3):269-280
14 Juul A.The effects of oestrogens on linear bone growth,Hum Reprod Update,2001,7:303-313
15 Pan W,Quarles LD,Xiao ZS,et al.Genistein stimulates the osteoblastic differentiation via NO-cGMP in bone marrow culture,J Cell Biochem, 2005,94(2):307-316
16刘敏,廖二元,周后德,等.17-β雌二醇对人成骨样细胞系MG 63 Cbfa 1表达的影响,中国骨质疏松杂志,2008,14(3):166-168
17郑碧琼,闫福华,骆凯,等.雌激素对正常及骨质疏松大鼠骨髓基质细胞增殖分化的影响,中国骨质疏松杂志,2007,13(1):22-25
18陈翠平,罗新.17β-雌二醇对大鼠骨髓间充质干细胞成骨分化的作用,中国骨质疏松杂志,2007,13(15):334-338
19 Hong L,Colpan A,Peptan IA.Modulations of 17-beta estradiol on osteogenic and adipogenic differentiations of human mesenchymal stem cells,Tissue Eng,2006
20赵玉岩,郭磊,都健,等.雌激素对成骨细胞增殖及IGF-2表达的调控作用,中国组织化学与细胞化学杂志,2008,17(4):311-314
21 Pufe T,Claassen H,Scholz-Ahrens KE,et al.Influence of estradiol on vascular endothelial growth factor expression in bone:a study in Gottingen miniature pigs and human osteoblasts,Calcif Tissue Int,2007,80 (3):184-191
22 Delhon I,Gutzwiller S,Toshimi Aizawa,et al.Absence of estrogenreceptor-related-alpha increases osteoblastic differentiation and cancellous bone mineral density,Endocrinology,2009,150(10):4463-4472
23 Hertrampf T,Schleipen B,Peng D,et al.Estrogen receptor subtype-specific effects on markers of bone homeostasis,Molecular and cellular endocrinology,2008,291(1-2):104-108
24 Bu YH,Peng D,Gutzwiller S,et al.Insulin receptor substrate 2 plays important roles in 17beta-estradiol-induced bone formation,Journal of endocrinological investigation,2009,32(8):682-689
2 Kharode YP,Sharp MC,Mary H,et al.Utility of the ovariectomizedratasa model for human osteoporosis in drug discovery,Methods in molecular biology,2008,45(5):111-124
3 Leitner MM,Tami AE,Hime M,et al.Longitudinal as well as age-matched assessments of bone changes in the mature ovariectomized rat model, Laboratory animals,2009,43(3):266-271
4 Srinivasan K,Ramarao P.Animal models in type 2 diabetes research:an overview,The Indian journal of medical research,2007,125(3):451-472
5 Unnanuntana A,Gladnick BP,Swift JM,et al.The assessment of fracture risk, The journal of bone and joint surgery American volume,2010,92(3):743-753
6 Kennedy OD,Brennan O,Ramarao P,et al.Effects of high bone turnover on the biomechanical properties of the L3 vertebra in an ovine model of early stage osteoporosis,Spine,2008,33(23):2518-2523
7 Kemper A,Ng T,Ma Y,et al.The biomechanical response of human bone: the influence of bone volume and mineral density. Biomedical sciences instrumentation,2006,42:284-289
8 Dai R,Ma Y,Kemper A,Prisby RD,et al.Effects of genistein on vertebral trabecular bone microstructure,bone mineral density,microcracks,osteocyte density,and bone strength in ovariectomized rats,Journal of bone and mineral metabolism,2008,26(4):342-349
9 Prisby RD,Swift JM,Franke S,et al.Altered bone mass,geometry and mechanical properties during the development and progression of type 2 diabetes in the Zucker diabetic fatty rat,J Endocrinol,2008,199(3):379-388
10刘亚平,季虹,荣海钦,等.2型糖尿病雌性大鼠骨代谢的特点,中国骨质疏松杂志,2008,14(1):35-38
11 Lipscombe LL,Jamal SA,Ma GZ,et al.The Risk of Hip Fractures in Older Individuals With Diabetes:A population-based study,diabetes care,2007, 30(4):835-841
12 Hanley DA,Tenenhouse A,Brennan O,et al.Associations among disease conditions,bone mineral density,and prevalent vertebral deformities in men and women 50 years of age and older:cross 2 sectional results from the Canadian Multicentre Osteoporosis Study,J Bone Miner Res,2003,18(4): 784-790
13 Hadjidakis DI,Androulakis II,Sommer M,et al.Diabetes in postmenopause: different influence on bone mass according to age and disease duration, Experimental and clinical endocrinology diabetes, 2009, 117(5):199-204
14 Franke S,Sommer M,Courtland HW,et al.Advanced glycation end products induce cell cycle arrest and proinflammatory changes in osteoarthritic fibroblast-like synovial cells,Arthritis research therapy,2009,1(5):R136
15 TakagiM,Kasayama S,Yamamo T,et al.A dvanced glycation end products stimulate interleukin-6 production by human bone-derived cells,Bone Miner Res,2003,12:439
16 Elis S,Courtland HW,Tenenhouse A,et al.Elevated serum levels of IGF-1 are sufficient to establish normal body size and skeletal properties,even in the absence of tissue IGF-1,Journal of bone and mineral research,2010,14:86-90
17 Kanazawal I,Yamaguchi T,Kasayama S,et al.Serum insulin-like growth factor-1 level is associated with the presence of vertebral fractures in postmenopausal women with type 2 diabetes mellitus,Osteoporosis international,2007,18(12):1675-1681
18秦健.2型糖尿病患者血糖控制前后对部分骨代谢指标、IGF-1及骨密度的影响,青岛医药卫生,2007,39(1):729
19黄昶荃,程刚.胰岛素抵抗对2型糖尿病及单纯胰岛素抵抗老龄大鼠的肾损伤和骨密度的影响,中国老年学杂志,2006,26:496-498
20 Chang-Quan H,Bi-Rong D,Swift JM,et al.Insufficient renal 1-alpha hydroxylase and bone homeostasis in aged rats with insulin resistance ortype 2 diabetes mellitus,Journal of bone and mineral metabolism, 2008, 26(6):561-568
21 Huang CQ,Ma GZ,Ma Y,et al.The relationship among renal injury, changed activity of trnal 1-alpha hydroxylase and bone loss in elderly rats with insulin resistance or type 2 diabetes mellitus. Journal of endocrinological investigation,2009,32(3):196-201
22 Magana JJ,Gomez R,Nabipour I,et al.Association of interleukin-6 gene polymorphisms with bone mineral density in Mexican women,Archives of medical research,2008,39(6):618-624
23 Li Y,Backesjo CM,Su Sc,et al.IL-6 receptor expression and IL-6 effects change during osteoblast differentiation,Cytokine,2008,43(2):165-173
24 Su Sc,Pei D,et al.Circulating pro-inflammatory cytokines and adiponectin in young men with type 2 diabetes,Acta diabetologica,2010,8:467-472
25 Xiang GD,Pu JH,Kono K,et al.Association between plasma osteoprotegerin concentrations and urinary albumin excretion in type 2 diabetes,Diabet med,2009,26(4):397-403
26 Nabipour I,Kalantarhormozi M,Courtland HW,et al.Osteoprotegerin in relation to type 2 diabetes mellitus and the metabolic syndrome in postmenopausal women,Metabolism clinical and experimental,2009, 17 (6):589-595
27 Fujii H,Kono K,Kasayama S,et al.Oxidative and Nitrosative Stress and Progression of Diabetic Nephropathy in type 2 diabetes,American journal of nephrology,2010,31(4):342-352
28 Schwartz AV,Sellmeyer DE,Strotmeyer ES,et al.Diabetes and bone loss at the hip in older black and white adults,J Bone Miner Res,2005,20(4):596-603
29 Janghorbani M,Van Dam RM,Willett WC,et al.Systematic review of type 1 and type 2 diabetes mellitus and risk of fracture,Am J Epidemiol, 2007,166(5):495-505
30 Strotmeyer ES,Cauley JA,Schwartz AV,et al.Diabetes is associated independently of body composition with BMD and bone volume in olderwhite and black men and women:The Health,Aging,and Body Composition Study,J Bone Miner Res,2004,19(7):1084-1091
31郝明,张瑜庆,延爱锦.老年2型糖尿病患者骨密度变化的探讨,山西医药杂志,2009,38(2):160-161
32李国娟,肖自幼,何皓,等.绝经后女性2型糖尿病患者骨密度变化及其相关因素探讨,实用糖尿病杂志,2008,4(2):34-35
33 Reinwald S,Peterson RG,Sellmeyer DE,et al.Skeletal changes associated with the onset of type 2 diabetes in the ZDF and ZDSD rodent models. American journal of physiology,endocrinology and metabolism, 2009,296(4):765-774
34张丽萍,荣海钦,季虹,等.2型糖尿病大鼠骨代谢和骨生物力学的观察,中国骨质疏松杂志,2008,14(5):318-323
1 Li Liang,Ji-feng Yu,Ken V,et al.Effect of estrogen receptor beta on the osteoblastic differentiation function of human periodontal ligament cells,archives of oral biology,2008,53(12):553–557
2李蔚,孙宜萍,朱国英,等.雌激素在体外对大鼠成骨细胞的作用,中国组织工程研究与临床康复,2008,12(11):2061-2064
3 Yan Zhou,Yun Fu,Lin Yang,et al.The role of estrogen in osteogenetic cytokine expression in human periodontal ligament cells,The international journal of periodontics restorative dentistry,2009,29(5):507-509
4 Sun J S,Li YY,Liu MH,et al.Effects of coumestrol on neonatal and adult mice osteoblast s activities,J Biomed Mater Res A,2007,81:214-223
5李蔚,孙宜萍,朱国英.雌激素在体外对大鼠成骨细胞的作用,中国组织工程研究与临床康复,2008,12(11):2061-2064
6武兆忠,刘敏,冯鉴强,等.雌激素膜快速效应对人成骨样细胞MG63OPGmRNA快速表达水平的影响,中国老年学杂志,2009,29 (10):1222-1224
7 Hertrampf T, Schleipen B, Velders M, et al.Estrogen receptor subtype-specific effects on markers of bone homeostasis,Mol cell endocrinol,2008,10,291(1-2):104-8
8 Pantschenko AG,Zhang W,Nahounou M,et al.Effect of osteoblast-targeted expression of bcl-2 in bone:differential response in male and female mice,J Bone Miner Res,2005,20(8):1414-1429
9 Peter G.Bradford,Ken V.Gerace,Toshimi Aizawa,et al. Estrogen regulation of apoptosis in osteoblasts,Physiology behavior,2010,99(56)181-185
10 Omar Khalid,Sanjeev K,Delhon I,et al.Modulation of Runx2 activity by Estrogen Receptorα:Implications for Osteoporosis and Breast Cancer,The Endocrine society,2008,23(6):11-13
11 Maruyama Z,Yoshida CA,Furuichi T,et al.Runx2 determines bonematurity and turnover rate in postnatal bone development and is involved in bone loss in estrogen deficiency,Dev Dyn,2007,236(7):1876-1890
12 Pufe T,Claassen H,Scholz-Ahrens KE,et al.Influence of estradiol on vascular endothelial growth factor expression in bone:a study in Gottingen miniature pigs and human osteoblasts,Calcif Tissue Int,2007, 80(3):184-191
13 Hiroyuki Takano,Toshimi Aizawa,Sanjeev K,et al.Normal bone growth requires optimal estrogen levels:negative effects of both high and low dose estrogen on the number of growth plate chondrocytes,Tohoku J Exp, 2008,214(3):269-280
14 Juul A.The effects of oestrogens on linear bone growth,Hum Reprod Update,2001,7:303-313
15 Pan W,Quarles LD,Xiao ZS,et al.Genistein stimulates the osteoblastic differentiation via NO-cGMP in bone marrow culture,J Cell Biochem, 2005,94(2):307-316
16刘敏,廖二元,周后德,等.17-β雌二醇对人成骨样细胞系MG 63 Cbfa 1表达的影响,中国骨质疏松杂志,2008,14(3):166-168
17郑碧琼,闫福华,骆凯,等.雌激素对正常及骨质疏松大鼠骨髓基质细胞增殖分化的影响,中国骨质疏松杂志,2007,13(1):22-25
18陈翠平,罗新.17β-雌二醇对大鼠骨髓间充质干细胞成骨分化的作用,中国骨质疏松杂志,2007,13(15):334-338
19 Hong L,Colpan A,Peptan IA.Modulations of 17-beta estradiol on osteogenic and adipogenic differentiations of human mesenchymal stem cells,Tissue Eng,2006
20赵玉岩,郭磊,都健,等.雌激素对成骨细胞增殖及IGF-2表达的调控作用,中国组织化学与细胞化学杂志,2008,17(4):311-314
21 Pufe T,Claassen H,Scholz-Ahrens KE,et al.Influence of estradiol on vascular endothelial growth factor expression in bone:a study in Gottingen miniature pigs and human osteoblasts,Calcif Tissue Int,2007,80 (3):184-191
22 Delhon I,Gutzwiller S,Toshimi Aizawa,et al.Absence of estrogenreceptor-related-alpha increases osteoblastic differentiation and cancellous bone mineral density,Endocrinology,2009,150(10):4463-4472
23 Hertrampf T,Schleipen B,Peng D,et al.Estrogen receptor subtype-specific effects on markers of bone homeostasis,Molecular and cellular endocrinology,2008,291(1-2):104-108
24 Bu YH,Peng D,Gutzwiller S,et al.Insulin receptor substrate 2 plays important roles in 17beta-estradiol-induced bone formation,Journal of endocrinological investigation,2009,32(8):682-689